The use of nucleic acid probe tests based on hybridization in routine clinical laboratory procedures is hindered by lack of sensitivity. The ability to amplify nucleic acids from clinical samples has greatly advanced nucleic acid probe technology, providing the sensitivity lacking in earlier versions of non-isotopic assays. Sensitivity afforded by oligonucleotide probe tests utilizing nucleic acid amplification now exceeds that of any other method. Nucleic acid amplification procedures can detect a single copy of a specific nucleic acid sequence. Routine detection and identification of specific gene sequences have extremely broad application in a number of settings and industries.
The major barrier for the transfer of technology to routine field testing is the absence of an economical and easy-to-use system or apparatus. In order to compete in today's cost conscious environment, genetic based testing must provide for high throughput, while incorporating adequate controls and safeguards to prevent false positive results due to sample cross-contamination.
Current technology involves several steps, although recent developments are directed toward automating systems for detection of the amplified target sequence. The first step, extraction of nucleic acids, is accomplished in a variety of ways, for example, phenol extraction, chaotropic reagent extraction, chromatographic purification (Qiagen, WO 95/01359, purification on silica membranes, specifically incorporated herein) and ultracentrifugation (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), specifically incorporated herein). Phenol is a well-established health hazard and requires special handling for waste removal. The extraction method is also tedious and labor intensive. Ultracentrifugation often requires the use of expensive and hazardous chemicals as well as the use of sophisticated and costly equipment. The process often requires long run times, sometimes involving one or more days of centrifugation. The easiest and fastest method is separation using chromatography purification.
The second step, the amplification of the target nucleic acid, employs a variety of enzymes known as polymerases and ligases. Polymerase chain reaction (PCR) is the most commonly used amplification technique. The general principles and conditions for amplification of nucleic acids using PCR are quite well known in the art; the details of which are provided in numerous references including U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,965,188, all to Mullis et al., all of which are specifically incorporated herein. Thus, the details of PCR technology are not included herein. Other approaches include ligase chain reaction, Q.beta. replicase, strand displacement assay, transcription mediated iso CR cycling probe technology and nucleic acid sequence-based amplification (NASBA).
A current protein detection technology for antigen-antibody assays involves the use of microparticles. Furthermore, a variety of microparticle strategies for dip-stick detection antigen-antibody assays are currently available, for example, a currently marketed at-home pregnancy test (U.S. Pat. No. 5,141,850 to Cole et al., specifically incorporated herein). Such tests use dyed particles that form a visible line following a specific antigen-antibody reaction. The instant invention is accomplished by hybridization of amplicons to capture oligonucleotides bound to microparticles. That is, the invention disclosed herein detects nucleic acid amplicons.
The detection of amplified nucleic acid for clinical use relies largely on hybridization of the amplified product and detection with a probe labeled with a variety of enzymes and luminescent reagents. U.S. Pat. No. 5,374,524 to Miller, specifically incorporated herein, describes a nucleic acid probe assay that combines nucleic acid amplification and solution hybridization using capture and reporter probes. These techniques require multiple reagents, several washing steps, and specialized equipment for detection of the target nucleic acid. Moreover, these techniques are labor intensive and require technicians with expertise in molecular biology.
The use of probes comprised of oligonucleotide sequences bound to microparticles is well known and illustrated in prior art. The mechanism for attachment of oligonucleotides to microparticles in hybridization assays and for the purification of nucleic acids is also well known. European Patent No. 200133, specifically incorporated herein, describes the attachment of oligonucleotides to water-insoluble particles less than 50 micrometers in diameter used in hybridization assays for the capture of target nucleotides. U.S. Pat. No. 5,387,512 to Wu, specifically incorporated herein, describes the use of oligonucleotide sequences covalently bound to microparticles as probes for capturing PCR amplified nucleic acids. U.S. Pat. No. 5,328,825 to Findlay, specifically incorporated herein, also describes an oligonucleotide linked by way of a protein or carbohydrate to a water-insoluble particle. The oligonucleotide probe is covalently coupled to the microparticle or other solid support. The sensitivity and specificity of all of the above-reference patents is based on hybridization of the oligonucleotide probe to the target nucleic acid.
The use of incorporated non-radioactive labels into the amplification reactions for the detection of nucleic acids is also well known in the art. Nucleic acids modified with biotin (U.S. Pat. No. 4,687,732 to Ward et al.; European Patent No. 063879; both specifically incorporated herein), digoxin (European Patent No. 173251, specifically incorporated herein) and other haptens have also been used. For example, U.S. Pat. No. 5,344,757 to Graf, specifically incorporated herein, uses a nucleic acid probe containing at least one hapten as label for hybridization with a complementary target nucleic acid bound to a solid membrane. The sensitivity and specificity of these assays is based on the incorporation of a single label in the amplification reaction which can be detected using an antibody specific to the label. The usual case involves an antibody conjugated to an enzyme. Furthermore, the addition of substrate generates a calorimetric or fluorescent change which can be detected with an instrument.
Still, the above-described approaches are labor intensive with many steps and washes; require special and costly equipment for the detection of the target nucleic acid; require trained staff; and take several hours to complete. Several patents have issued which deal with automation of the processes of amplification and subsequent detection of the amplicon. These patents use specialized equipment and are still based on the principle of hybridization and immunoassay technology. For example, European Patent No. 320308, specifically incorporated herein, describes a system detecting target nucleic acids amplified by the ligase chain reaction.
Automated approaches eliminate the need for specially trained personnel, however, the cost of the equipment is very high and the possibility of contamination still exists since many samples will be processed by the same equipment. To eliminate the issue of contamination, it is necessary to integrate the three steps outlined above. The self-contained device disclosed herein accomplishes this goal by integrating existing nucleic acid extraction and isothermal amplification technologies with an innovative detection strategy.
The invention described herein provides for the rapid and accurate detection of amplified nucleic acid sequences using a self-contained device. The possibility of contamination is eliminated because of the "throw away" approach described herein. Elimination of cross contamination opens the door to mass screening including automation. The high sensitivity of the analysis allows for the early detection of disease and an opportunity for early treatment. The present invention diagnoses the presence of infectious diseases of genetic, bacterial or viral origin. Analysis by this invention can monitor the efficacy of treatment, for example, to monitor HIV virus in the plasma of patients undergoing therapy. Analysis, according to the invention disclosed herein, is easy, requiring little expertise in the art of molecular biology. The cost is significantly less than other methods currently in use to detect amplified nucleic acids. The time frame for detecting an amplified sequence is reduced drastically. There is no danger from potentially hazardous chemicals. The analysis does not require special waste disposal procedures. The requirements of many washes in an immunometric or hybridization approach are eliminated. The self-contained device does not require special equipment, other than a standard, constant temperature heat block. The low complexity of the device lends itself to "point of care" testing in clinics and physician's offices. The portability of the device provides for "on site" analysis to detect nucleic acid sequences in the areas of forensics, agriculture, environment and the food industry.
Nucleic acid probe technology has developed rapidly in recent years as the scientific community has discovered its value for detection of various diseases, organisms or genetic abnormalities. Amplification techniques have provided the sensitivity to qualitatively determine the presence of even minute quantities of nucleic acid. The drawback to wide spread use of this technology is the possibility of cross contamination of samples since the test is so sensitive. The cost of nucleic acid based testing is high as it requires highly skilled technicians and sophisticated equipment. One method of eliminating the possibility of carry over from one sample to another, is to use a completely enclosed disposable device.